Transglutaminases are protective enzymes which are responsible for blood clotting in response to tissue injury under normal conditions. However, these enzymes are also reported to play a crucial role in the pathological mechanism of various diseases in the absence of regulatory control of the level of expression thereof (Soo-Youl Kim: New Target Against Inflammatory Diseases: Transglutaminase 2. Archivum Immunologiae & Therapiae Experimentalis 52, 332-337, 2004. Soo-Youl Kim. Transglutaminase 2 in inflammation. Front Biosci. 11, 3026-3035, 2006). The expression of transglutaminases increases particularly upon the occurrence of various inflammatory diseases, including rheumatoid arthritis, diabetes, inflammatory myositis, atherosclerosis, stroke, liver cirrhosis, breast cancer, Alzheimer's disease, Parkinson's disease, Huntington's disease, encephalitis, celiac disease, etc. Also, transglutaminases are observed to increase in expression level, along with NF-κB, when cancer enters metastasis or changes into chemo-resistance or radio-resistance (Soo-Youl Kim. Transglutaminase 2 in inflammation. Front Biosci. 11, 3026-3035, 2006).
The relationship between transglutaminases and chemo-resistance in cancer has remained unclear thus far. However, when the expression of transglutaminases was suppressed in chemoresistant breast cancer cells, the cancer cells were getting highly susceptible to chemicals, and finally died (Antonyak et al., Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells. J Biol Chem. 2004 Oct. 1;279(40):41461-7.; Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and NF-κB Inactivation. Cancer Res. 2006. 66, 10936-10943).
Also, there is a strong reason for suppressing the activity of transglutaminases as the etiological mechanism for which the activation of transglutaminases is responsible is elucidated at the molecular level (Key Chung Park, Kyung Cheon Chung, Yoon-Seong Kim, Jongmin Lee, Tong H. Joh, and Soo-Youl Kim. Transglutaminase 2 induces nitric oxide synthesis in BV-2 microglia. Biochem. Biophys. Res. Commun. 323, 1055-1062, 2004; Jongmin Lee, Yoon-Seong Kim, Dong-Hee Choi, Moon S. Bang, Tay R. Han, Tong H. Joh, and Soo-Youl Kim. Transglutaminase 2 induces NF-KB activation via a novel pathway in BV-2 microglia. J. Biol. Chem. 279, 53725-53735, 2004; Dae-Seok Kim et al. Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-KB Inactivation. Cancer Res. 2006. Cancer Res. 2006. 66, 10936-10943).
Inflammation is largely attributable to NF-κB activation. NF-κB is known to be activated by kinases in signal transduction pathways. However, NF-κB was also found to be activated independently of kinases, thereby negating the function of kinase inhibitors (Tergaonkar et al., I-kappaB kinase-independent I-κBα degradation pathway: functional NF-kappaB activity and implications for cancer therapy. Mol Cell Biol. 2003 Nov;23(22):8070-83.).
In a previous study conducted by the present inventors, it was reported that transglutaminase activates NF-κB independently of the activation of kinases (IKK, NAK), by inducing crosslinking I-κBα (Jongmin Lee, et al. Transglutaminase 2 induces NF-κB activation via a novel pathway in BV-2 microglia. J. Biol. Chem. 279, 53725-53735, 2004). Transglutaminases are calcium-dependent enzymes, which can activate NF-κB only at an elevated intracellular level of calcium.
Upon inflammation, the activation of the transcriptional factor NF-κB leads to an increase in the expression not only of inflammatory factors including transglutaminases, but also of its inhibitor I-κBα. Continuous NF-κB activation is inhibited by I-κBα under normal conditions, but continues in chronic inflammatory diseases. Interestingly, TNF-α or LPS (lipopolysaccharide)-induced NF-κB activation gives rise to transglutaminase expression. Thus, aberrantly activated transglutaminases in inflammatory cells are expected to activate NF-κB directly or to further maintain activated NF-κB, thereby playing a key role in inflammation maintenance (FIG. 1). In addition, this vicious cycle may be a main cause of cancer metastasis and chemo-resistance (Jongmin Lee, et al. Transglutaminase 2 induces NF-kB activation via a novel pathway in BV-2 microglia. J. Biol. Chem. 279, 53725-53735, 2004).
Therefore, a transglutaminase inhibitor may play a crucial role in breaking the continuous cycle of NF-κB, on which the steroid-substituting effect proposed by the present inventors is based (Sohn, J., Kim, T. I., Yoon, Y. H., and Kim, S. Y.: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis. J. Clin. Invest. 111, 121-8, 2003).
Amine compounds are known to inhibit transglutaminase activity. Representative of the TGase inhibitors are cystamine (Nature Genetics 18, 111-117, 1998; Nature Medicine 8, 143-149, 2002) and putrescine. In addition to the amine compounds, other chemical inhibitors, such as monodansylcadaverine (J. Med. Chem. 15, 674-675, 1972), w-dibenzylaminoalkylamine (J. Med. Chem. 18, 278-284, 1975), 3-halo-4,5-dihydroisoxazole (Mol. Pharmacol. 35, 701-706, 1989), and 2-[(2-oxopropyl)thio]imidazolium derivatives (Blood, 75, 1455-1459, 1990), were developed, but are reported to be so toxic as to non-specifically inhibit other enzymes in vivo.
Therefore, there is a need for safe and effective transglutaminase-specific inhibitors. Recently, Sohn et al. have succeeded in obtaining the same effect from recombinant peptides as steroidal drugs for the inflammation of allergic conjunctivitis to ragweed in a guinea pig model (Sohn, J., Kim, T. -I., Yoon, Y. -H., and Kim, S. -Y.: Transglutaminase Inhibitor: A New Anti-Inflammatory Approach in Allergic Conjunctivitis. J. Clin. Invest. 111, 121-8, 2003). In this regard, anti-flammin protein (PLA2 inhibitor)- or elafin protein (very strong transglutaminase substrate, Nara, K., et al. 1994. Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed “cementoin”. J Biochem (Tokyo). 115:441-448)-derived synthetic peptides which mimic the catalytic site of transglutaminase were used.
Leading to the present invention, intensive and thorough research based on the foregoing, conducted by the present inventors, allows the present inventors to identify the positions of the glutamine and lysine residues involved in the transglutaminase-induced I-κBα polymerization, and find that I-κBα-derived peptides or homologs thereof containing these residues inhibit transglutaminase-induced I-κBα polymerization, thus acting as transglutaminase inhibitors.